Method for producing oxo fatty acid and rare fatty acid

ABSTRACT

The present invention provides a production method of oxo fatty acid, as well as rare fatty acids such as conjugated fatty acid, hydroxylated fatty acid, partially saturated fatty acid and the like, which uses 4 kinds of enzymes (fatty acid-hydratase, hydroxylated fatty acid-dehydrogenase, oxo fatty acid-isomerase, oxo fatty acid-enone reductase) derived from Lactobacillus plantarum including lactic acid bacteria and the like. Furthermore, the present invention also provides a more efficient production method of oxo fatty acid and the like, which partly uses a chemical oxidation reaction in combination.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a divisional of U.S. patent application Ser.No. 14/400,116, filed on Nov. 10, 2014, now U.S. Pat. No. 9,719,115,which is the U.S. national phase of International Patent Application No.PCT/JP2012/078747, filed Nov. 6, 2012, which claims the benefit ofJapanese Patent Application No. 2012-108928, filed on May 10, 2012,which are incorporated by reference in their entireties herein.

INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY SUBMITTED

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: 6,274 bytes ASCII (Text) file named“729082SequenceListing.txt,” created Jun. 19, 2017.

TECHNICAL FIELD

The present invention relates to a production method of a fatty acid.More detailedly, the present invention relates to a production method ofan oxo fatty acid, comprising using an unsaturated fatty acid as astarting material and multi-step enzyme reaction or combining a chemicaloxidation reaction and an enzyme reaction method, and a productionmethod of a rare fatty acid from an oxo fatty acid.

BACKGROUND ART

Conjugated fatty acid represented by conjugated linoleic acid (CLA) hasbeen reported to have various physiological activities such as a lipidmetabolism improving effect, an anti-arteriosclerosis action, a bodyfats decreasing action and the like (non-patent documents 1-3), and is afunctional lipid expected to be applicable to various fields ofmedicament, functional food and the like (patent documents 1, 2). WhileCLA is known to be contained in dairy products and meat products sinceit is produced by microorganisms present in the stomach of ruminant andto be present in a small amount in vegetable oil, the detailed mechanismof production thereof is not known.

The present inventors reported that 3 kinds of enzymes present in thefungus body of Lactobacillus plantarum (CLA-HY, CLA-DC, CLA-DH) areessential for the reaction to convert linoleic acid to conjugatedlinoleic acid (patent document 1). However, the mechanism of a series ofspecific reactions, the presence of an intermediate and the like inthese enzyme reactions have not been clarified.

In addition, it has been reported in recent years that oxo fatty acidssuch as 9-oxo-octadecadienoic acid, 13-oxo-octadecadienoic acid and thelike contained in tomato have an activity to improve lifestyle-relateddiseases, such as lipid metabolism improvement and the like (patentdocument 3, non-patent documents 4, 5), and the physiological activityof oxo fatty acid is drawing attention. While oxo fatty acid has acarbonyl group at a particular position of unsaturated fatty acid,synthesis of functional oxo fatty acid from unsaturated fatty acid isdifficult since it is necessary to distinguish double bonds present in aplurality in a molecule and introduce carbonyl group into a particularposition. Also, it is not known that rare fatty acids such as CLA1, CLA2and the like are produced from a particular oxo fatty acid.

DOCUMENT LIST Patent Documents

-   patent document 1: JP-A-2007-259712-   patent document 2: JP-A-2007-252333-   patent document 3: JP-A-2011-184411

Non-Patent Documents

-   non-patent document 1: Ha Y L, (1987), Carcinogenesis, vol. 8, no.    12, p. 1881-1887-   non-patent document 2: Clement Ip, (1991), Cancer Res., (1991), vol.    51, p. 6118-6124-   non-patent document 3: Kisun N L, (1994), Atherosclerosis, vol.    108, p. 19-25-   non-patent document 4: Kim Y-I, (2011), Mol. Nutr. Food Res., vol.    55, p. 585-593-   non-patent document 5: Kim Y-I, (2012), PLoS ONE, vol. 7, no. 2,    e31317

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

An object of the present invention is to provide a method of efficientlyproducing an oxo fatty acid, and a method of producing a rare fatty acidsuch as hydroxylated fatty acid, conjugated fatty acid, partiallysaturated fatty acid and the like from the produced oxo fatty acid.

Means of Solving the Problems

The present inventors have clarified the full particulars of theunsaturated fatty acid metabolic pathway of lactic acid bacteria, foundoxo fatty acid, hydroxylated fatty acid, conjugated fatty acid andpartially saturated fatty acid as intermediates for the metabolicsystem, and identified a novel enzyme (CLA-ER) involved in theproduction of them.

To be specific, using known enzymes (CLA-HY, CLA-DC, CLA-DH) and a novelenzyme (CLA-ER), the present inventors have clarified a series ofmechanisms of the production of cis-9, trans-11-conjugated linoleic acid(c9,t11-CLA (CLA1)), trans-9, trans-11-conjugated linoleic acid(t9,t11-CLA (CLA2)), oleic acid, trans-10-octadecenoic acid (t10-18:1)and the like from linoleic acid (see FIGURE). They have also found thatoxo fatty acids such as 10-oxo-cis-12-octadecenoic acid (hereinafter tobe also referred to as “KetoA”), 10-oxooctadecanoic acid (hereinafter tobe also referred to as “KetoB”), 10-oxo-trans-11-octadecenoic acid(hereinafter to be also referred to as “KetoC”) and the like areproduced as intermediates for the reaction, and further that theconversion efficiency is remarkably improved by introducing a chemicaloxidation method using chromic acid instead of a part of enzymereactions (oxidation reaction of hydroxylated fatty acid).

In addition, the present inventors have also found that, using oxo fattyacid, large supply of which has been achieved for the first time by thepresent invention as a starting material, rare fatty acids such ashydroxylated fatty acid, conjugated fatty acid and partially saturatedfatty acid can be produced by a conventionally-unknown reaction pathwayof Keto C from Keto A, Keto B from Keto C,10-hydroxy-trans-11-octadecenoic acid from Keto C (hereinafter to bealso referred to as “HYC”), 10-hydroxy-octadecanoic acid from Keto B(hereinafter to be also referred to as “HYB”), CLA1 or CLA2 from HYC,oleic acid or trans-10-octadecenoic acid from HYB, and linoleic acid ortrans-10,cis-12-conjugated linoleic acid (t10, c12-CLA (CLA3)) from10-hydroxy-cis-12-octadecenoic acid (hereinafter to be also referred toas “HYA”). The present invention was completed based on the abovefindings.

Accordingly, the present invention provides the following:

[1] A method of producing an oxo fatty acid having 18 carbon atoms and acarbonyl group at the 10-position, comprising inducing a hydroxylatedfatty acid having 18 carbon atoms and a hydroxyl group at the10-position from an unsaturated fatty acid having 18 carbon atoms and acis-type double bond at the 9-position by a hydratase reaction, andsubjecting the hydroxylated fatty acid to a dehydrogenase reaction orchemical oxidation.[2] The method of [1], wherein the unsaturated fatty acid having 18carbon atoms and a cis-type double bond at the 9-position is oleic acid,linoleic acid, γ-linolenic acid, α-linolenic acid, stearidonic acid,cis-9,trans-11-octadecadienoic acid or ricinoleic acid.[3] The method of [1] or [2], wherein the hydratase and thedehydrogenase are derived from lactic acid bacteria.[4] The method of [3], wherein the lactic acid bacteria is Lactobacillusplantarum FERM BP-10549 strain.[5] A method of producing an oxo fatty acid having 18 carbon atoms, acarbonyl group at the 10-position and a trans-type double bond at the11-position from an oxo fatty acid having 18 carbon atoms, a carbonylgroup at the 10-position and a cis-type double bond at the 12-position,by an isomerase reaction.[6] The method of [5], wherein the oxo fatty acid having 18 carbonatoms, a carbonyl group at the 10-position and a cis-type double bond atthe 12-position is 10-oxo-cis-12-octadecenoic acid,10-oxo-cis-6,cis-12-octadecadienoic acid,10-oxo-cis-12,cis-15-octadecadienoic acid or10-oxo-cis-6,cis-12,cis-15-octadecatrienoic acid.[7] The method of [5] or [6], wherein the isomerase is derived fromlactic acid bacteria.[8] The method of [7], wherein the lactic acid bacteria is Lactobacillusplantarum FERM BP-10549 strain.[9] A method of producing an oxo fatty acid having 18 carbon atoms and acarbonyl group at the 10-position, and not having a double bond at the11- and 12-positions from an oxo fatty acid having 18 carbon atoms, acarbonyl group at the 10-position and a trans-type double bond at the11-position by a saturase.[10] The method of [9], wherein the oxo fatty acid having 18 carbonatoms, a carbonyl group at the 10-position and a trans-type double bondat the 11-position is 10-oxo-trans-11-octadecenoic acid,10-oxo-cis-6,trans-11-octadecadienoic acid,10-oxo-trans-11,cis-15-octadecadienoic acid or10-oxo-cis-6,trans-11,cis-15-octadecatrienoic acid.[11] The method of [9] or [10], wherein the saturase is derived fromlactic acid bacteria.[12] The method of [11], wherein the lactic acid bacteria isLactobacillus plantarum FERM BP-10549 strain.[13] An enzyme protein of any of the following (a)-(c):(a) an enzyme protein consisting of the amino acid sequence shown in SEQID NO: 2,(b) a protein comprising an amino acid sequence which is the amino acidsequence shown in SEQ ID NO: 2 wherein one or plural amino acids aredeleted and/or substituted and/or inserted and/or added, and having anenzyme activity of catalyzing the saturation reaction in [9],(c) a protein encoded by a base sequence that hybridizes to a nucleicacid consisting of a complementary chain sequence of the base sequenceshown in SEQ ID NO: 1 under stringent conditions, and having an enzymeactivity to catalyze the saturation reaction in [9].[14] A nucleic acid encoding the enzyme protein of [13].[15] A vector comprising the nucleic acid of [14].[16] A host cell transformed with the vector of [15].[17] A method of producing an enzyme, comprising culturing the host cellof [16], and recovering the enzyme protein of [13] from the culture.[18] The method of [9], wherein the saturase is the protein of [13].[19] A method of producing a hydroxylated fatty acid having 18 carbonatoms, a hydroxyl group at the 10-position and a trans-type double bondat the 11-position from an oxo fatty acid having 18 carbon atoms, acarbonyl group at the 10-position and a trans-type double bond at the11-position, by a dehydrogenase reaction.[20] The method of [19], wherein the oxo fatty acid having 18 carbonatoms, a carbonyl group at the 10-position and a trans-type double bondat the 11-position is 10-oxo-trans-11-octadecenoic acid,10-oxo-cis-6,trans-11-octadecadienoic acid,10-oxo-trans-11,cis-15-octadecadienoic acid or10-oxo-cis-6,trans-11,cis-15-octadecatrienoic acid.[21] The method of [19] or [20], wherein the dehydrogenase is derivedfrom lactic acid bacteria.[22] The method of [21], wherein the lactic acid bacteria isLactobacillus plantarum FERM BP-10549 strain.[23] A method of producing a hydroxylated fatty acid having 18 carbonatoms and a hydroxyl group at the 10-position, and not having a doublebond at the 11- and 12-positions from an oxo fatty acid having 18 carbonatoms and a carbonyl group at the 10-position and not having a doublebond at the 11- and 12-positions, by a dehydrogenase reaction.[24] The method of [23], wherein the oxo fatty acid having 18 carbonatoms and a carbonyl group at the 10-position and not having a doublebond at the 11- and 12-positions is 10-oxooctadecanoic acid,10-oxo-cis-6-octadecenoic acid, 10-oxo-cis-15-octadecenoic acid or10-oxo-cis-6,cis-15-octadecadienoic acid.[25] The method of [23] or [24], wherein the dehydrogenase is derivedfrom lactic acid bacteria.[26] The method of [25], wherein the lactic acid bacteria isLactobacillus plantarum FERM BP-10549 strain.[27] A method of producing a conjugated fatty acid having a cis-typedouble bond at the 9-position and a trans-type double bond at the11-position or a conjugated fatty acid having a trans-type double bondat the 9- and 11-positions from a hydroxylated fatty acid having 18carbon atoms, a hydroxyl group at the 10-position and a trans-typedouble bond at the 11-position by a dehydratase reaction.[28] The method of [27], wherein the hydroxylated fatty acid having 18carbon atoms, a hydroxyl group at the 10-position and a trans-typedouble bond at the 11-position is 10-hydroxy-trans-11-octadecenoic acid,10-hydroxy-cis-6,trans-11-octadecadienoic acid,10-hydroxy-trans-11,cis-15-octadecadienoic acid or10-hydroxy-cis-6,trans-11,cis-15-octadecatrienoic acid.[29] The method of [27] or [28], wherein the dehydratase is derived fromlactic acid bacteria.[30] The method of [29], wherein the lactic acid bacteria isLactobacillus plantarum FERM BP-10549 strain.[31] A method of producing a partially saturated fatty acid having acis-type double bond at the 9-position or a partially saturated fattyacid having a trans-type double bond at the 10-position from ahydroxylated fatty acid having 18 carbon atoms and a hydroxyl group atthe 10-position, and not having a double bond at the 11- and12-positions by a dehydratase reaction.[32] The method of [31], wherein the hydroxylated fatty acid having 18carbon atoms and a hydroxyl group at the 10-position, and not having adouble bond at the 11- and 12-positions is 10-hydroxyoctadecanoic acid,10-hydroxy-cis-6-octadecenoic acid, 10-hydroxy-cis-15-octadecenoic acidor 10-hydroxy-cis-6,cis-15-octadecadienoic acid.[33] The method of [31] or [32], wherein the dehydratase is derived fromlactic acid bacteria.[34] The method of [33], wherein the lactic acid bacteria isLactobacillus plantarum FERM BP-10549 strain.[35] A method of producing a conjugated fatty acid having a cis-typedouble bond at the 9- and 12-positions or a conjugated fatty acid havinga trans-type double bond at the 10-position and a cis-type double bondat the 12-position from a hydroxylated fatty acid having 18 carbonatoms, a hydroxyl group at the 10-position and a cis-type double bond atthe 12-position by a dehydratase reaction.[36] The method of [35], wherein the hydroxylated fatty acid having 18carbon atoms, a hydroxyl group at the 10-position and a cis-type doublebond at the 12-position is 10-hydroxy-cis-12-octadecenoic acid,10-hydroxy-cis-6,cis-12-octadecadienoic acid,10-hydroxy-cis-12,cis-15-octadecadienoic acid or10-hydroxy-cis-6,cis-12,cis-15-octadecatrienoic acid.[37] The method of [35] or [36], wherein the dehydratase is derived fromlactic acid bacteria.[38] The method of [37], wherein the lactic acid bacteria isLactobacillus plantarum FERM BP-10549 strain.

Effect of the Invention

In the present invention, a conventionally-unknown saturase (CLA-ER) wasfound and combined with known enzymes, based on which the fullparticulars of the unsaturated fatty acid metabolic pathway of lacticacid bacteria was clarified. Furthermore, a multi-step enzyme reactioncan produce an oxo fatty acid, a more efficient conversion can beperformed by changing a part of said reaction to a chemical oxidationreaction, and oxo fatty acid can be produced in a large amount. Also,rare fatty acid can be efficiently produced from oxo fatty acid, and theoxo fatty acid, rare fatty acid and the like are extremely useful sincethey can be used in various fields of medicament, food, cosmetic and thelike.

BRIEF DESCRIPTION OF THE DRAWINGS

The FIGURE shows the whole image of the production method of the oxofatty acid and rare fatty acid of the present invention, whereinlinoleic acid was used as a starting material.

DESCRIPTION OF EMBODIMENTS

The present invention is explained in detail below.

The present invention provides a method of producing useful rare fattyacids such as oxo fatty acid, hydroxylated fatty acid, conjugated fattyacid, partially saturated fatty acid and the like, which comprisesperforming each reaction of the unsaturated fatty acid metabolismpathway of lactic acid bacteria by enzyme methods (and chemically wherenecessary for a reaction with low enzyme reaction efficiency) in anappropriate combination. One embodiment of an overall reaction system isshown in the FIGURE.

The first aspect of the present invention provides a method of producingan oxo fatty acid having 18 carbon atoms and a carbonyl group at the10-position (hereinafter sometimes to be abbreviated as “10-oxo fattyacid”) from an unsaturated fatty acid having 18 carbon atoms and acis-type double bond at the 9-position (hereinafter sometimes to beabbreviated as “cis-9 unsaturated fatty acid”) by two-step reaction. Inthe first reaction (reaction 1), a hydroxylated fatty acid having 18carbon atoms and a hydroxyl group at the 10-position (hereinaftersometimes to be abbreviated as “10-hydroxyfatty acid”) is produced fromcis-9 unsaturated fatty acid by a hydratase reaction.

The substrate in “reaction 1” is not particularly limited as long as itis an unsaturated fatty acid having 18 carbon atoms and a cis-typedouble bond at the 9-position, and examples thereof include monoeneoicacid (18:1), dienoic acid (18:2), trienoic acid (18:3), tetraenoic acid(18:4), pentaenoic acid (18:5) and the like. More preferred are dienoicacid, trienoic acid and tetraenoic acid, and particularly preferred aredienoic acids and trienoic acids. In the present specification, “fattyacid” encompasses not only free acids but also ester form, salt withbasic compound and the like.

Examples of the monoenoic acid include oleic acid, ricinoleic acid andthe like.

Examples of the dienoic acid include linoleic acid (cis-9,cis-12-18:2),cis-9,trans-11-octadecadienoic acid (cis-9,trans-11-18:2) and the like.

Examples of the trienoic acids include α-linolenic acid(cis-9,cis-12,cis-15-18:3), γ-linolenic acid (cis-6,cis-9,cis-12-18:3)and the like.

Examples of the tetraenoic acid include stearidonic acid(cis-6,cis-9,cis-12,cis-15-18:4) and the like.

While hydratase that catalyzes reaction 1 is not particularly limited aslong as it is an enzyme capable of utilizing the above-mentioned cis-9unsaturated fatty acid as a substrate and capable of converting to10-hydroxyfatty acid, for example, lactic acid bacteria-derived fattyacid-hydratase (CLA-HY) is preferable. More preferred is Lactobacillusplantarum-derived CLA-HY, and particularly preferred is L. plantarumFERM BP-10549 strain-derived CLA-HY. CLA-HY can be obtained by themethod described in JP-A-2007-259712, or the method described in thebelow-mentioned Examples. Hydratase may be a purified one or a crudelypurified one. Alternatively, hydratase may be expressed in fungus suchas Escherichia coli and the like and the fungus itself may be used orculture medium thereof may be used. Furthermore, the enzyme may be of afree type, or immobilized by various carriers.

The hydratase reaction may be performed in a suitable buffer (e.g.,phosphate buffer, tris buffer, borate buffer etc.) by mixing cis-9unsaturated fatty acid, which is a substrate, and hydratase at suitableconcentrations and incubating the mixture. The substrate concentrationis, for example, 1-100 g/L, preferably 10-50 g/L, more preferably 20-40g/L. The amount of hydratase to be added is, for example, 0.001-10mg/ml, preferably 0.1-5 mg/ml, more preferably 0.2-2 mg/ml.

A “cofactor” may be used for reaction 1 and, for example, NADH, NADPH,FADH₂ and the like can be used. The concentration of addition may be anyas long as the hydration reaction proceeds efficiently. It is preferably0.001-20 mM, more preferably 0.01-10 mM.

Furthermore, an “activator” may be used for the enzyme reaction and, forexample, one or more compounds selected from the group consisting ofpotassium molybdate, disodium molybdate(VI) anhydrate, disodiummolybdate(VI) dihydrate, sodium orthovanadate(V), sodiummetavanadate(V), potassium tungstate(VI), sodium tungstate(VI) anhydrateand sodium tungstate(VI) dihydrate can be mentioned. The concentrationof addition thereof may be any as long as the hydration reactionproceeds efficiently. It is preferably 0.1-20 mM, more preferably 1-10mM.

Reaction 1 is desirably performed within the ranges of preferabletemperature and preferable pH of hydratase. For example, the reactiontemperature is 5-50° C., preferably 20-45° C. The pH of the reactionmixture is pH 4-10, preferably pH 5-9. The reaction time is notparticularly limited and it is, for example, 10 min-72 hr, preferably 30min-36 hr.

In one preferable one embodiment of the present invention, hydratase isprovided to the reaction system in the form of recombinant cells (e.g.,Escherichia coli, Bacillus subtilis, yeast, insect cell, animal celletc.) introduced with an expression vector containing a nucleic acidencoding same. In this case, the hydratase reaction can also beperformed by cultivating the cells in a liquid medium suitable for theculture of the cells and added with a substrate and, where necessary, acofactor and an activator.

In the second reaction (reaction 2) of the first aspect of the presentinvention, an oxo fatty acid having 18 carbon atoms and a carbonyl groupat the 10-position (hereinafter sometimes to be abbreviated as “10-oxofatty acid”) is produced from 10-hydroxyfatty acid by a dehydrogenasereaction or chemical oxidation using chromic acid.

While the dehydrogenase that catalyzes reaction 2 is not particularlylimited as long as it is an enzyme capable of utilizing 10-hydroxyfattyacid as a substrate and capable of converting to 10-oxo fatty acid, forexample, lactic acid bacteria-derived hydroxylated fattyacid-dehydrogenase (CLA-DH) is preferable. More preferred isLactobacillus plantarum-derived CLA-DH, and particularly preferred is L.plantarum FERM BP-10549 strain-derived CLA-DH. CLA-DH can be obtained bythe method described in JP-A-2007-259712, or the method described in thebelow-mentioned Examples. Dehydrogenase may be a purified one or acrudely purified one. Alternatively, dehydrogenase may be expressed infungus such as Escherichia coli and the like and the fungus itself maybe used or culture medium thereof may be used. Furthermore, the enzymemay be of a free type, or immobilized by various carriers.

The dehydrogenase reaction may be performed in a suitable buffer (e.g.,phosphate buffer, tris buffer, borate buffer etc.) by mixing10-hydroxyfatty acid, which is a substrate, and dehydrogenase atsuitable concentrations and incubating the mixture. The substrateconcentration is, for example, 1-100 g/L, preferably 10-50 g/L, morepreferably 20-40 g/L. The amount of dehydrogenase to be added is, forexample, 0.001-10 mg/ml, preferably 0.1-5 mg/ml, more preferably 0.2-2mg/ml.

A “cofactor” may be used for reaction 2 and, for example, NAD, NADP, FADand the like can be used. The concentration of addition may be any aslong as the oxidation reaction proceeds efficiently. It is preferably0.001-20 mM, more preferably 0.01-10 mM.

Furthermore, an “activator” may be used for the enzyme reaction and, forexample, compounds similar to those recited as examples in theabove-mentioned reaction 1 can be used at a similar additionconcentration.

Reaction 2 is desirably performed within the ranges of preferabletemperature and preferable pH of dehydrogenase. For example, thereaction temperature is 5-50° C., preferably 20-45° C. The pH of thereaction mixture is, for example, pH 4-10, preferably pH 5-9. Thereaction time is not particularly limited and it is, for example, 10min-72 hr, preferably 30 min-36 hr.

In one embodiment of the present invention, dehydrogenase is provided tothe reaction system in the form of recombinant cells (e.g., Escherichiacoli, Bacillus subtilis, yeast, insect cell, animal cell etc.)introduced with an expression vector containing a nucleic acid encodingsame. In this case, the oxidation reaction can also be performed bycultivating the cells in a liquid medium suitable for the culture of thecells and added with a substrate and, where necessary, a cofactor and anactivator.

As shown in the below-mentioned Examples 9 and 11, the present inventorshave found that the conversion efficiency of 10-hydroxyfatty acid to10-oxo fatty acid is comparatively low when L. plantarum FERM BP-10549strain-derived CLA-DH is used as dehydrogenase. Therefore, they changedreaction 2 to a chemical oxidation using chromic acid, whereby extremelyhigh conversion efficiency could be successfully obtained. Accordingly,in the first aspect of the present invention, the second reaction ismore preferably performed by chemical oxidation.

As the chemical oxidation, methods known per se, for example, chromicacid oxidation, preferably Jones oxidation and the like can bementioned. As the chromic acid, salts and complexes of the compound suchas anhydrous chromic acid CrO₃, chromic acid H₂CrO₄ and dichromic acidH₂Cr₂O₇ can be used.

The second aspect of the present invention provides a method ofproducing an oxo fatty acid having 18 carbon atoms, a carbonyl group atthe 10-position and a trans-type double bond at the 11-position(hereinafter sometimes to be abbreviated as “10-oxo,trans-11 fattyacid”) from an oxo fatty acid having 18 carbon atoms, a carbonyl groupat the 10-position and a cis-type double bond at the 12-position(hereinafter sometimes to be abbreviated as “10-oxo,cis-12 fatty acid”)by an isomerase reaction (reaction 3).

The “substrate” of reaction 3 is not particularly limited as long as itis 10-oxo,cis-12 fatty acid induced from an unsaturated fatty acidhaving 18 carbon atoms and a cis-type double bond at the 9- and12-positions, by the above-mentioned reactions 1 and 2. Examples thereofinclude 10-oxo-cis-12-octadecenoic acid (KetoA) induced from linoleicacid, 10-oxo-cis-12,cis-15-octadecadienoic acid (αKetoA) induced fromα-linolenic acid, 10-oxo-cis-6,cis-12-octadecadienoic acid (γKetoA)induced from γ-linolenic acid, 10-oxo-cis-6, cis-12,cis-15-octadecatrienoic acid (sKetoA) induced from stearidonic acid andthe like. It is needless to say that the substrate may be obtained by amethod other than reactions 1 and 2.

While isomerase that catalyzes reaction 3 is not particularly limited aslong as it is an enzyme capable of utilizing the above-mentioned10-oxo,cis-12 fatty acid as a substrate and capable of converting to10-oxo,trans-11 fatty acid, for example, lactic acid bacteria-derivedoxo fatty acid-isomerase (CLA-DC) is preferable. More preferred isLactobacillus plantarum-derived CLA-DC, and particularly preferred is L.plantarum FERM BP-10549 strain-derived CLA-DC. CLA-DC can be obtained bythe method described in JP-A-2007-259712, or the method described in thebelow-mentioned Examples. Isomerase may be a purified one or a crudelypurified one. Alternatively, isomerase may be expressed in fungus suchas Escherichia coli and the like and the fungus itself may be used orculture medium thereof may be used. Furthermore, the enzyme may be of afree type, or immobilized by various carriers.

The isomerase reaction may be performed in a suitable buffer (e.g.,phosphate buffer, tris buffer, borate buffer etc.) by mixing10-oxo,cis-12 fatty acid, which is a substrate, and isomerase atsuitable concentrations and incubating the mixture. The substrateconcentration is, for example, 1-100 g/L, preferably 10-50 g/L, morepreferably 20-40 g/L. The amount of isomerase to be added is, forexample, 0.001-10 mg/ml, preferably 0.1-5 mg/ml, more preferably 0.2-2mg/ml.

An “activator” may be used for the isomerase reaction and, for example,compounds similar to those recited as examples in the above-mentionedreaction 1 can be used at a similar addition concentration.

Reaction 3 is desirably performed within the ranges of preferabletemperature and preferable pH of isomerase. For example, the reactiontemperature is 5-50° C., preferably 20-45° C. The pH of the reactionmixture is, for example, pH 4-10, preferably pH 5-9. The reaction timeis not particularly limited and it is, for example, 10 min-72 hr,preferably 30 min-36 hr.

In one preferable embodiment of the present invention, isomerase isprovided to the reaction system in the form of recombinant cells (e.g.,Escherichia coli, Bacillus subtilis, yeast, insect cell, animal celletc.) introduced with an expression vector containing a nucleic acidencoding same. In this case, the isomerase reaction can also beperformed by cultivating the cells in a liquid medium suitable for theculture of the cells and added with a substrate and, where necessary, anactivator.

The third aspect of the present invention provides a method of producingan oxo fatty acid having 18 carbon atoms and a carbonyl group at the10-position and not having a double bond at the 11- and 12-positions(hereinafter sometimes to be abbreviated as “10-oxo,11,12-saturatedfatty acid”) from an oxo fatty acid having 18 carbon atoms, a carbonylgroup at the 10-position and a trans-type double bond at the 11-position(10-oxo,trans-11 fatty acid) by a saturase reaction (reaction 4).

The “substrate” of reaction 4 is not particularly limited as long as itis 10-oxo,trans-11 fatty acid produced by the above-mentioned reaction3. Examples thereof include 10-oxo-trans-11-octadecenoic acid (KetoC)induced from 10-oxo-cis-12-octadecenoic acid (KetoA),10-oxo-trans-11,cis-15-octadecadienoic acid (to be also referred to as“αKetoC”) induced from 10-oxo-cis-12,cis-15-octadecadienoic acid (to bealso referred to as “αKetoA”), 10-oxo-cis-6,trans-11-octadecadienoicacid (to be also referred to as “γKetoC”) induced from10-oxo-cis-6,cis-12-octadecadienoic acid (to be also referred to as“γKetoA”), 10-oxo-cis-6,trans-11,cis-15-octadecatrienoic acid (to bealso referred to as “sKetoC”) induced from10-oxo-cis-6,cis-12,cis-15-octadecatrienoic acid (to be also referred toas “sKetoA”) and the like. It is needless to say that the substrate maybe obtained by a method other than reaction 3.

While saturase that catalyzes reaction 4 is not particularly limited aslong as it is an enzyme capable of utilizing the above-mentioned10-oxo,trans-11 fatty acid as a substrate and capable of converting to10-oxo,11,12-saturated fatty acid, for example, oxo fatty acid-enonereductase (CLA-ER) derived from lactic acid bacteria isolated in thepresent invention is preferable. More preferred is Lactobacillusplantarum-derived CLA-ER, and particularly preferred is L. plantarumFERM BP-10549 strain-derived CLA-ER.

The novel enzyme “CLA-ER” of the present invention is

(a) an enzyme protein consisting of the amino acid sequence shown in SEQID NO: 2,

(b) a protein comprising an amino acid sequence which is the amino acidsequence shown in SEQ ID NO: 2 wherein one or plural amino acids aredeleted and/or substituted and/or inserted and/or added, and having anenzyme activity of catalyzing the above-mentioned reaction 4, or(c) a protein encoded by a base sequence that hybridizes to a nucleicacid consisting of a complementary chain sequence of the base sequenceshown in SEQ ID NO: 1 under stringent conditions, and having an enzymeactivity to catalyze the above-mentioned reaction 4.

More specific examples of the above-mentioned (b) include a proteincontaining (i) an amino acid sequence which is the amino acid sequenceshown in SEQ ID NO: 2, wherein 1-20, preferably 1-10, more preferably1-several (5, 4, 3 or 2) amino acids are deleted, (ii) an amino acidsequence which is the amino acid sequence shown in SEQ ID NO: 2, wherein1-20, preferably 1-10, more preferably 1-several number (5, 4, 3 or 2)amino acids are added, (iii) an amino acid sequence which is the aminoacid sequence shown in SEQ ID NO: 2, wherein 1-20, preferably 1-10, morepreferably 1-several (5, 4, 3 or 2) amino acids are inserted, (iv) anamino acid sequence which is the amino acid sequence shown in SEQ ID NO:2, wherein 1-20, preferably 1-10, more preferably 1-several (5, 4, 3 or2) amino acids are substituted by other amino acids, or (v) an aminoacid sequence obtained by combining them. When amino acids with similarproperties (e.g., glycine and alanine, valine and leucine andisoleucine, serine and threonine, aspartic acid and glutamic acid,asparagine and glutamine, lysin and arginine, cysteine and methionine,phenylalanine and tyrosine etc.) are substituted with each other and thelike, a greater number of substitutions and the like are possible.

When amino acids are deleted, substituted or inserted as mentionedabove, the positions of deletion, substitution and insertion are notparticularly limited as long as the above-mentioned enzyme activity ismaintained.

In the above-mentioned (c), the “stringent conditions” are conditionsunder which nucleotide sequences having high identity, for example,identity of 70, 80, 90, 95 or 99% or above, hybridize to each other andnucleotide sequences having identity lower than that do not hybridize;specifically, conditions of washing once, more preferably 2-3 times, atthe salt concentration and temperature corresponding to those in thewashing conditions of general Southern hybridization (60° C., 1×SSC,0.1% SDS, preferably, 0.1×SSC, 0.1% SDS, more preferably, 68° C.,0.1×SSC, 0.1% SDS) and the like.

CLA-ER can be isolated from, for example, the fungus and culture mediumof L. plantarum FERM BP-10549 strain by a protein separation andpurification technique known per se. Alternatively, CLA-ER can also beproduced as a recombinant protein by isolating a gene encoding CLA-ERaccording to the method described in Example 2, subcloning same into asuitable vector, introducing same into a suitable host such asEscherichia coli and the like and culturing same. CLA-ER may be apurified one or a crudely purified one. Alternatively, hydratase may beexpressed in fungus such as Escherichia coli and the like and the fungusitself may be used or culture medium thereof may be used. Furthermore,the enzyme may be of a free type, or immobilized by various carriers.

As a vector containing a nucleic acid encoding CLA-ER of the presentinvention, one suitable for a host cell to be introduced with the vectormay be appropriately selected according to the object (e.g., proteinexpression) and can be used. In the case of an expression vector, itcontains the nucleic acid of the present invention, which is operablylinked to an appropriate promoter, and preferably contains atranscription termination signal, i.e., terminator region, at thedownstream of the nucleic acid of the present invention. Furthermore, itcan also contain a selection marker gene for selection of a transformant(drug resistance gene, gene that complements auxotrophic mutation etc.).Also, it may contain a sequence encoding a tag sequence useful forseparation and purification of the expressed protein and the like. Inaddition, the vector may be incorporated into the genome of a targethost cell. The vector of the present invention can be introduced into atarget host cell by a transformation method known per se such as acompetent cell method, a protoplast method, a calcium phosphatecoprecipitation method and the like.

In the present invention, the “host cell” may be any cell as long as itcan express a vector containing a nucleic acid encoding CLA-ER of thepresent invention, and bacterium, yeast, fungi, higher eukaryotic celland the like can be mentioned. Examples of the bacterium includegram-positive bacteria such as bacillus, Streptomyces and the like andgram negative bacteria such as Escherichia coli and the like. Arecombinant cell introduced with a vector containing a nucleic acidencoding CLA-ER can be cultivated by a method known per se which issuitable for the host cell.

In the present invention, “purification” of CLA-ER can be performed by amethod known per se, for example, fungi collected by centrifugation andthe like are ruptured by ultrasonication or glass beads and the like,solid such as cell debris is removed by centrifugation and the like, andthe like to give a crude enzyme solution, which is subjected to asalting out method using ammonium sulfate, sodium sulfate and the like,chromatographys such as ion exchange chromatography, gel filtrationchromatography, affinity chromatography and the like, gelelectrophoresis and the like.

The isomerase reaction may be performed in a suitable buffer (e.g.,phosphate buffer, tris buffer, borate buffer etc.) by mixing10-oxo,trans-11 fatty acid, which is a substrate, and saturase atsuitable concentrations and incubating the mixture. The substrateconcentration is, for example, 1-100 g/L, preferably 10-50 g/L, morepreferably 20-40 g/L. The amount of saturase to be added is, forexample, 0.001-10 mg/ml, preferably 0.1-5 mg/ml, more preferably 0.2-2mg/ml.

A “cofactor” may be used for reaction 4 and, for example, NADH and thelike can be used. The concentration of addition may be any as long asthe oxidation reaction proceeds efficiently. It is preferably 0.001-20mM, more preferably 0.01-10 mM.

Furthermore, an “activator” may be used for the enzyme reaction and, forexample, compounds similar to those recited as examples in theabove-mentioned reaction 1 can be used at a similar additionconcentration.

Reaction 4 is desirably performed within the ranges of preferabletemperature and preferable pH of saturase. For example, the reactiontemperature is 5-50° C., preferably 20-45° C. The pH of the reactionmixture is, for example, pH 4-10, preferably pH 5-9. The reaction timeis not particularly limited and it is, for example, 10 min-72 hr,preferably 30 min-36 hr.

In one preferable embodiment of the present invention, saturase isprovided to the reaction system in the form of recombinant cells (e.g.,Escherichia coli, Bacillus subtilis, yeast, insect cell, animal celletc.) introduced with an expression vector containing a nucleic acidencoding same. In this case, the saturase reaction can also be performedby cultivating the cells in a liquid medium suitable for the culture ofthe cells and added with a substrate and, where necessary, an activator.

The fourth aspect of the present invention provides a method ofproducing a hydroxylated fatty acid having 18 carbon atoms, a hydroxylgroup at the 10-position and a trans-type double bond at the 11-position(hereinafter sometimes to be abbreviated as “10-hydroxy,trans-11 fattyacid”) from an oxo fatty acid having 18 carbon atoms, a carbonyl groupat the 10-position and a trans-type double bond at the 11-position(10-oxo,trans-11 fatty acid) by a dehydrogenase reaction (reaction 5) ora method of producing a hydroxylated fatty acid having 18 carbon atomsand a hydroxyl group at the 10-position, and not having a double bond atthe 11- and 12-positions (hereinafter sometimes to be abbreviated as“10-hydroxy,11,12-saturated fatty acid”) from an oxo fatty acid having18 carbon atoms and a carbonyl group at the 10-position and not having adouble bond at the 11- and 12-positions (10-oxo,11,12-saturated fattyacid) by a dehydrogenase reaction (reaction 6).

The “substrate” of reaction 5 is not particularly limited as long as itis 10-oxo,trans-11 fatty acid produced by the above-mentioned reaction3. Examples thereof include 10-oxo-trans-11-octadecenoic acid (KetoC)induced from 10-oxo-cis-12-octadecenoic acid (KetoA),10-oxo-trans-11,cis-15-octadecadienoic acid (to be also referred to as“αKetoC”) induced from 10-oxo-cis-12,cis-15-octadecadienoic acid (to bealso referred to as “αKetoA”), 10-oxo-cis-6,trans-11-octadecadienoicacid (to be also referred to as “γKetoC”) induced from10-oxo-cis-6,cis-12-octadecadienoic acid (to be also referred to as“γKetoA”), 10-oxo-cis-6,trans-11,cis-15-octadecatrienoic acid (to bealso referred to as “sKetoC”) induced from10-oxo-cis-6,cis-12,cis-15-octadecatrienoic acid (to be also referred toas “sKetoA”) and the like. It is needless to say that the substrate maybe obtained by a method other than reaction 3.

On the other hand, the “substrate” of reaction 6 is not particularlylimited as long as it is 10-oxo,11,12-saturated fatty acid produced bythe above-mentioned reaction 4. Examples thereof include10-oxooctadecanoic acid (KetoB) induced from10-oxo-trans-11-octadecenoic acid (KetoC), 10-oxo-cis-15-octadecenoicacid (to be also referred to as “αKetoB”) induced from10-oxo-trans-11,cis-15-octadecadienoic acid (αKetoC),10-oxo-cis-6-octadecenoic acid (to be also referred to as “γKetoB”)induced from 10-oxo-cis-6,trans-11-octadecadienoic acid (γKetoC),10-oxo-cis-6,cis-15-octadecadienoic acid (to be also referred to as“sKetoB”) induced from 10-oxo-cis-6,trans-11,cis-15-octadecatrienoicacid (sKetoC) and the like. It is needless to say that the substrate maybe obtained by a method other than reaction 4.

While the dehydrogenase that catalyzes reaction 5 or reaction 6 is notparticularly limited as long as it is an enzyme capable of utilizing10-oxo,trans-11 fatty acid or 10-oxo,11,12-saturated fatty acid as asubstrate and capable of converting to 10-hydroxy,trans-11 fatty acid or10-hydroxy,11,12-saturated fatty acid, for example, lactic acidbacteria-derived hydroxylated fatty acid-dehydrogenase (CLA-DH) ispreferable. More preferred is Lactobacillus plantarum-derived CLA-DH,and particularly preferred is L. plantarum FERM BP-10549 strain-derivedCLA-DH. While CLA-DH catalyzes the oxidation reaction in theabove-mentioned reaction 2, it can also catalyze the reduction reactionin reaction 5 or reaction 6 as a reverse reaction.

Dehydrogenase may be a purified one or a crudely purified one.Alternatively, dehydrogenase may be expressed in fungus such asEscherichia coli and the like and the fungus itself may be used orculture medium thereof may be used. Furthermore, the enzyme may be of afree type, or immobilized by various carriers.

The reduction reaction by dehydrogenase may be performed in a suitablebuffer (e.g., phosphate buffer, tris buffer, borate buffer etc.) bymixing 10-oxo,trans-11 fatty acid or 10-oxo,11,12-saturated fatty acid,which is a substrate, and dehydrogenase at suitable concentrations andincubating the mixture. The substrate concentration is, for example,1-100 g/L, preferably 10-50 g/L, more preferably 20-40 g/L. The amountof dehydrogenase to be added is, for example, 0.001-10 mg/ml, preferably0.1-5 mg/ml, more preferably 0.2-2 mg/ml.

A “cofactor” may be used for reaction 5 and reaction 6 and, for example,NADH, NADPH, FADH₂ and the like can be used. The concentration ofaddition may be any as long as the reduction reaction proceedsefficiently. It is preferably 0.001-20 mM, more preferably 0.01-10 mM.

Furthermore, an “activator” may be used for the enzyme reaction and, forexample, compounds similar to those recited as examples in theabove-mentioned reaction 1 can be used at a similar additionconcentration.

Reaction 5 and reaction 6 are desirably performed within the ranges ofpreferable temperature and preferable pH of dehydrogenase. For example,the reaction temperature is 5-50° C., preferably 20-45° C. The pH of thereaction mixture is, for example, pH 4-10, preferably pH 5-9. Thereaction time is not particularly limited and it is, for example, 10min-72 hr, preferably 30 min-36 hr.

In one preferable one embodiment of the present invention, dehydrogenaseis provided to the reaction system in the form of recombinant cells(e.g., Escherichia coli, Bacillus subtilis, yeast, insect cell, animalcell etc.) introduced with an expression vector containing a nucleicacid encoding same. In this case, the reduction reaction can also beperformed by cultivating the cells in a liquid medium suitable for theculture of the cells and added with a substrate and, where necessary, acofactor and an activator.

The fifth aspect of the present invention provides a method of producinga conjugated fatty acid having a cis-type double bond at the 9-positionand a trans-type double bond at the 11-position (hereinafter sometimesto be abbreviated as “cis-9,trans-11 conjugated fatty acid”) or aconjugated fatty acid having a trans-type double bond at the 9- and11-positions (hereinafter sometimes to be abbreviated as“trans-9,trans-11 conjugated fatty acid”) from a hydroxylated fatty acidhaving 18 carbon atoms, a hydroxyl group at the 10-position and atrans-type double bond at the 11-position (10-hydroxy,trans-11 fattyacid) by a dehydratase reaction (reaction 7), a method of producing apartially saturated fatty acid having a cis-type double bond at the9-position (hereinafter sometimes to be abbreviated as “cis-9 partiallysaturated fatty acid”) or a partially saturated fatty acid having atrans-type double bond at the 10-position (hereinafter sometimes to beabbreviated as “trans-10 partially saturated fatty acid”) from ahydroxylated fatty acid having 18 carbon atoms and a hydroxyl group atthe 10-position, and not having a double bond at the 11- and12-positions (10-hydroxy,11,12-saturated fatty acid) by a dehydratasereaction (reaction 8), or a method of producing a unsaturated fatty acidhaving a cis-type double bond at the 9- and 12-positions (hereinaftersometimes to be abbreviated as “cis-9,cis-12 unsaturated fatty acid”) ora conjugated fatty acid having a trans-type double bond at the10-position and a cis-type double bond at the 12-position (hereinaftersometimes to be abbreviated as “trans-10,cis-12 conjugated fatty acid”)from a hydroxylated fatty acid having 18 carbon atoms, a hydroxyl groupat the 10-position and a cis-type double bond at the 12-position(hereinafter sometimes to be abbreviated as “10-hydroxy,cis-12 fattyacid”) by a dehydratase reaction (reaction 9).

The “substrate” of reaction 7 is not particularly limited as long as itis 10-hydroxy,trans-11 fatty acid produced by the above-mentionedreaction 5. Examples thereof include 10-hydroxy-trans-11-octadecenoicacid (HYC) induced from 10-oxo-trans-11-octadecenoic acid (KetoC),10-hydroxy-trans-11,cis-15-octadecadienoic acid (to be also referred toas “αHYC”) induced from 10-oxo-trans-11,cis-15-octadecadienoic acid(αKetoC), 10-hydroxy-cis-6,trans-11-octadecadienoic acid (to be alsoreferred to as “γHYC”) induced from10-oxo-cis-6,trans-11-octadecadienoic acid (γKetoC),10-hydroxy-cis-6,trans-11,cis-15-octadecatrienoic acid (to be alsoreferred to as “sHYC”) induced from10-oxo-cis-6,trans-11,cis-15-octadecatrienoic acid (sKetoC) and thelike. It is needless to say that the substrate may be obtained by amethod other than reaction 5.

The “substrate” of reaction 8 is not particularly limited as long as itis 10-hydroxy,11,12-saturated fatty acid produced by the above-mentionedreaction 6. Examples thereof include 10-hydroxyoctadecanoic acid (HYB)induced from 10-oxooctadecanoic acid (KetoB),10-hydroxy-cis-15-octadecenoic acid (to be also referred to as “αHYB”)induced from 10-oxo-cis-15-octadecenoic acid (αKetoB),10-hydroxy-cis-6-octadecenoic acid (to be also referred to as “γHYB”)induced from 10-oxo-cis-6-octadecenoic acid (γKetoB),10-hydroxy-cis-6,cis-15-octadecadienoic acid (to be also referred to as“sHYB”) induced from 10-oxo-cis-6,cis-15-octadecadienoic acid (sKetoB)and the like. It is needless to say that the substrate may be obtainedby a method other than reaction 6.

The “substrate” of reaction 9 is not particularly limited as long as itis 10-hydroxy,cis-12 fatty acid that can be produced from an unsaturatedfatty acid having a cis-type double bond at the 9- and 12-positions bythe above-mentioned reaction 1. Examples thereof include10-hydroxy-cis-12-octadecenoic acid (HYA) induced from linoleic acid,10-hydroxy-cis-12,cis-15-octadecadienoic acid (to be also referred to as“αHYA”) induced from α-linolenic acid,10-hydroxy-cis-6,cis-12-octadecadienoic acid (to be also referred to as“γHYA”) induced from γ-linolenic acid,10-hydroxy-cis-6,cis-12,cis-15-octadecatrienoic acid (to be alsoreferred to as “sHYA”) induced from stearidonic acid and the like. It isneedless to say that the substrate may be obtained by a method otherthan reaction 1.

While hydratase that catalyzes reactions 7-9 is not particularly limitedas long as it is an enzyme capable of utilizing the above-mentioned10-hydroxy,trans-11 fatty acid, 10-hydroxy,11,12-saturated fatty acid or10-hydroxy,cis-12 fatty acid as a substrate and capable of converting tocis-9,trans-11 conjugated fatty acid or trans-9,trans-11 conjugatedfatty acid, cis-9 partially saturated fatty acid or trans-10 partiallysaturated fatty acid, or cis-9,cis-12 unsaturated fatty acid ortrans-10,cis-12 conjugated fatty acid, for example, lactic acidbacteria-derived fatty acid-hydratase (CLA-HY) is preferable. Morepreferred is Lactobacillus plantarum-derived CLA-HY, and particularlypreferred is L. plantarum FERM BP-10549 strain-derived CLA-HY. WhileCLA-HY catalyzes the hydration reaction in the above-mentioned reaction1, it can also catalyze the dehydration reaction in reactions 7-9 as areverse reaction.

Dehydratase may be a purified one or a crudely purified one.Alternatively, hydratase may be expressed in fungus such as Escherichiacoli and the like and the fungus itself may be used or culture mediumthereof may be used. Furthermore, the enzyme may be of a free type, orimmobilized by various carriers.

The dehydratase reaction may be performed in a suitable buffer (e.g.,phosphate buffer, tris buffer, borate buffer etc.) by mixing10-hydroxy,trans-11 fatty acid, 10-hydroxy,trans-11,12-saturated fattyacid or 10-hydroxy,cis-12 fatty acid, which is a substrate, anddehydratase at suitable concentrations and incubating the mixture. Thesubstrate concentration is, for example, 1-100 g/L, preferably 10-50g/L, more preferably 20-40 g/L. The amount of dehydratase to be addedis, for example, 0.001-10 mg/ml, preferably 0.1-5 mg/ml, more preferably0.2-2 mg/ml.

A “cofactor” may be used for reactions 7-9 and, for example, NADH,NADPH, FADH₂ and the like can be used. The concentration of addition maybe any as long as the dehydration reaction proceeds efficiently. It ispreferably 0.001-20 mM, more preferably 0.01-10 mM.

Furthermore, an “activator” may be used for the enzyme reaction and, forexample, compounds similar to those recited as examples in theabove-mentioned reaction 1 can be used at a similar additionconcentration.

Reactions 7-9 are desirably performed within the ranges of preferabletemperature and preferable pH of dehydratase. For example, the reactiontemperature is 5-50° C., preferably 20-45° C. The pH of the reactionmixture is, for example, pH 4-10, preferably pH 5-9. The reaction timeis not particularly limited and it is, for example, 10 min-72 hr,preferably 30 min-36 hr.

In one preferable embodiment of the present invention, dehydratase isprovided to the reaction system in the form of recombinant cells (e.g.,Escherichia coli, Bacillus subtilis, yeast, insect cell, animal celletc.) introduced with an expression vector containing a nucleic acidencoding same. In this case, the dehydration reaction can also beperformed by cultivating the cells in a liquid medium suitable for theculture of the cells and added with a substrate and, where necessary, acofactor and an activator.

The oxo fatty acid, hydroxylated fatty acid, conjugated fatty acid orpartially saturated fatty acid (hereinafter to be comprehensivelyreferred to as “oxo fatty acid and the like”) obtained in the presentinvention can be used by being blended with, for example, medicament,food or cosmetic agent based on the conventionally-known physiologicalactivity.

Examples of the dosage form of a medicament containing oxo fatty acidand the like include powder, granule, pill, soft capsule, hard capsules,tablet, chewable tablet, quick-integrating tablet, syrup, liquid,suspension, suppository, ointment, cream, gel, adhesive, inhalant,injection and the like. A preparation thereof is prepared according to aconventional method. Since oxo fatty acid and the like are poorlysoluble in water, they are dissolved in a non-hydrophilic organicsolvent such as plant-derived oil, animal-derived oil and the like ordispersed or emulsified in an aqueous solution together with anemulsifier, a dispersing agent, a surfactant and the like by ahomogenizer (high-pressure homogenizer) and used.

Examples of the additives that can be used for formulating includeanimal and plant oils such as soybean oil, safflower oil, olive oil,germ oil, sunflower oil, beef fat, sardine oil and the like, polyvalentalcohols such as polyethylene glycol, propylene glycol, glycerol,sorbitol and the like, surfactants such as sorbitan ester of fatty acid,sucrose fatty acid ester, glycerin fatty acid ester, polyglycerin fattyacid ester and the like, excipients such as purified water, lactose,starch, crystalline cellulose, D-mannitol, lecithin, gum arabic,sorbitol solution, carbohydrate solution and the like, sweetener,colorant, pH adjuster, flavor and the like. A liquid preparation may bedissolved or suspended in water or other suitable medium when in use.Also, tablet and granules may be coated by a well-known method.

For administration in the form of an injection, intravenous,intraperitoneal, intramuscular, subcutaneous, transdermal,intraarticular, intrasynovial, intrathecal, intraperiosteum, sublingual,oral administrations and the like are preferable, and intravenousadministration or intraperitoneal administration is particularlypreferable. The intravenous administration may be any of dripadministration and bolus administration.

Examples of the form of the “food” containing oxo fatty acid and thelike obtained by the present invention include supplements (powder,granule, soft capsule, hard capsule, tablet, chewable tablet,quick-integrating tablet, syrup, liquid etc.), drinks (tea, carbonicacid drink, lactic acid drink, sport drink etc.), confectionery (gummy,jelly, gum, chocolate, cookie, candy etc.), oil, fat and oil food(mayonnaise, dressing, butter, cream, margarine etc.) and the like.

The above-mentioned foods can contain, where necessary, variousnutrients, various vitamins (vitamin A, vitamin B1, vitamin B2, vitaminB6, vitamin C, vitamin D, vitamin E, vitamin K etc.), various minerals(magnesium, zinc, iron, sodium, potassium, selenium etc.), dietaryfiber, dispersing agent, stabilizer such as emulsifier and the like,sweetener, flavor components (citric acid, malic acid etc.), flavor,royal jelly, propolis, Agaricus and the like.

Examples of the “cosmetic agent” containing oxo fatty acid and the likeobtained by the present invention include cream, skin milk, toner,microemulsion essence, bath powder and the like, which may be mixed witha flavor and the like.

The present invention is explained in more detail in the following byreferring to Examples. The Examples are mere exemplifications of thepresent invention and do not limit the scope of the present invention inany manner.

Example 1

Culture Method of Lactobacillus plantarum FERM BP-10549

Lactobacillus plantarum FERM BP-10549 in MRS stab containing 2% agar andpreserved at 4° C. was inoculated in 15 ml of MRS liquid medium(manufactured by Difco; pH 6.5), and precultured at 28° C., 120 rpm for20 hr. The main culture was performed by culturing the total amount ofthe preculture, inoculated to 550 ml of MRS liquid medium containing 7.7ml of linoleic acid solution shown below, at 28° C., 120 rpm for 24 hr.The linoleic acid solution was obtained by adding 10 mg of bovine serumalbumin to 50 mg of linoleic acid, suspending same in 1 ml of 0.1 Mpotassium phosphate buffer (pH 6.5), homogenizing the suspension byultrasonication for 10 min, and eliminating bacteria by using a 0.45 μmfilter. After culture, centrifugation at 3,000 rpm, 4° C. for 10 mingave the fungus of Lactobacillus plantarum FERM BP-10549.

Lactobacillus plantarum FERM BP-10549 strain has been deposited sinceMar. 7, 2006 at incorporated administrative agency, International PatentOrganism Depositary of National Institute of Advanced Industrial Scienceand Technology (IPOD, AIST) (now incorporated administrative agency,International Patent Organism Depositary of National Institute ofTechnology and Evaluation (IPOD, NITE)); Central 6, 1-1-1 Higashi,Tsukuba, Ibaraki 305-8566 Japan.

Example 2

Cloning of Gene of Enzyme Having Amino Acid Sequence Shown in SEQ ID NO:2 (CLA-ER:Oxo Fatty Acid-Enone Reductase)

(1) Obtainment of Genome DNA

Wet fungus (14 g) of Lactobacillus plantarum FERM BP-10549 was suspendedin 180 ml of TEN buffer (10 mM Tris-HCl (pH 7.5); 1 mM EDTA; 10 mMNaCl). Thereto were added 9 ml of SET buffer (20% Sucrose; 50 mM EDTA;50 mM Tris-HCl (pH 7.5)) and 135 mg of lysozyme and the mixture wasincubated at 37° C. for 10 min. Then, 90 ml of TEN buffer, 9 ml of 25%SDS, 18 ml of 5 M NaCl, 180 ml of phenol, and 32 ml of chloroform wereadded and the mixture was gently and completely mixed. Thereafter, themixture was centrifuged at 3,500×g for 20 min at room temperature, andthe upper layer was recovered. Then, chloroform in an equal amount tothe upper layer was added, the mixture was completely mixed andcentrifuged at 3,500×g for 20 min at room temperature, and the upperlayer was recovered. An equal amount of ethanol was added, the mixturewas completely mixed and centrifuged at 3,500×g for 20 min at roomtemperature. The obtained precipitate was dried for 20 min in a vacuumdesiccator, and dissolved in a small amount of TE buffer (10 mM Tris-HCl(pH 8.0); 1 mM EDTA). Thereto was added 20 μl of RNaseA solution, themixture was incubated at 37° C. for 15 hr, 1.2 ml of chloroform wasadded, the mixture was centrifuged at 3,500×g for 20 min at roomtemperature and the upper layer was recovered. Thereto was added 6 ml ofchloroform, the mixture was incubated at 3,500×g for 20 min at roomtemperature, and the upper layer was recovered. Furthermore, 6 ml ofisopropanol was added, the mixture was completely mixed, incubated for30 min at room temperature and centrifuged at 3,500×g for 20 min at roomtemperature. The obtained precipitate was washed with 70% ethanol, driedin a vacuum desiccator, and dissolved in TE buffer to give genome DNA.

(2) Obtainment of CLA-ER Gene by PCR

In the published full-length genome gene sequence of Lactobacillusplantarum WCFS1 strain, an open-reading-frame (ORF) present immediatelyat the downstream of CLA-DC was used as a target. A sense primer (SEQ IDNO: 3) was designed based on the sequence at 9-28 bases upstream of the5′ side of the initiation codon of ORF, and an antisense primer (SEQ IDNO: 4) was designed based on the sequence 13-31 bases downstream of the3′ side of the stop codon. Using these primers, and the genome DNA ofLactobacillus plantarum FERM BP-10549 as a template, PCR was performed.The base sequence of about 0.7 kbp gene segment amplified as a result ofPCR was decoded. As a result, it was clarified that the gene segmentcontains one 654 bp ORF (SEQ ID NO: 1), which starts from the initiationcodon ATG and ends at the stop codon TAA, and this gene was termed asCLA-ER gene. The CLA-ER gene encodes a protein consisting of 217residual amino acids shown in SEQ ID NO: 2.

Example 3

Expression of (CLA-ER) in Escherichia coli

A host vector system consisting of Escherichia coli expression vectorpET101/D-TOPO (Invitrogen) and Rosetta 2 (DE3) strain was used. CACC wasadded to the upstream of the initiation codon ATG of CLA-ER gene ofLactobacillus plantarum FERM BP-10549, and PCR was performed using asense primer (SEQ ID NO: 5) designed based on the initiation codon andthe sequence of 23 bases on the 3′ terminal side containing same, and anantisense primer (SEQ ID NO: 6) designed based on the sequence of 26bases on the 5′ terminal side from which the stop codon TAA had beendeleted, and the genome DNA of Lactobacillus plantarum FERM BP-10549 asa template. An about 0.6 kbp gene segment amplified as a result of PCRwas inserted into pET101/D-TOPO to construct an expression vector(pCLA-ER). Rosetta 2 (DE3) strain was transformed with pCLA-ER to givetransformed Rosetta/pCLA-ER strain. The obtained Rosetta/pCLA-ER strainwas aerobically cultured in 10 ml LB medium (medium containing 1% BactoTripton (Difco), 0.5% yeast extract, 1% sodium chloride (pH 7.0))containing 1 mg ampicillin at 37° C., 300 rpm for 15 hr to give apreculture. The preculture (10 ml) was inoculated to 750 ml LB mediumcontaining 75 mg ampicillin, aerobically cultured at 37° C., 100 rpm for2 hr. Then, 1 M IPTG (750 μl) was added, and the mixture was aerobicallyfurther cultured at 20° C., 100 rpm for 15 hr. After culture, themixture was centrifuged at 3,000 rpm for 10 min to give wet fungus ofRosetta/pCLA-ER strain. As saturase, CLA-ER expressing transformedEscherichia coli was used.

Example 4

Preparation Method of Each Transformed Escherichia coli

Based on the report of Kishino et al. (Biochemical and BiophysicalResearch Communications 416 (2011) 188-193), transformed Escherichiacoli expressing each of CLA-HY, CLA-DH, CLA-DC was prepared. Ashydratase and dehydratase, CLA-HY expression transformed Escherichiacoli was used, CLA-DC expressing transformed Escherichia coli was usedas isomerase and CLA-DH expressing transformed Escherichia coli was usedas dehydrogenase.

Example 5

Purification of Dehydratase from Dehydratase Transformed Escherichiacoli

The fungus obtained by culture was suspended in 3 ml of buffer C (40 mMimidazole, 50 mM potassium phosphate buffer (pH 8.0), 0.5 M NaCl), andthe fungus was disrupted by ultrasonication. After disruption, thefungus was ultracentrifuged at 10,000×g, 4° C., for 60 min and the upperlayer was recovered and purified by FPLC using affinity column (HisTrapHP) and dialysis (dialysis solution was 50 mM potassium phosphate buffer(pH 6.5)).

Example 6

Purification of Dehydrogenase from Dehydrogenase Transformed Escherichiacoli

The fungus obtained by culture was suspended in 3 ml of BugBuster MasterMix (Novagen), and the fungus was incubated at room temperature for 20min. After incubation, the fungus was ultracentrifuged at 10,000×g, 4°C., for 60 min and the upper layer was recovered and purified by FPLCusing gel filtration column (Superdex 200 Hiload 26/60), ion exchangecolumn (MonoQ 10/100), gel filtration column (Superdex 200 10/300) anddialysis (dialysis solution was buffer C).

Example 7

Production of HYA from Linoleic Acid by Using Hydratase Expressed inEscherichia coli

Using hydratase-induced transformed Escherichia coli, a production testof HYA from linoleic acid was performed. The reaction mixture was 100 mMpotassium phosphate buffer (pH 6.5) containing hydratase-inducedtransformed Escherichia coli (wet fungus weight 3 g), NADH (600 mg), FAD(15 mg), linoleic acid (5 g) and BSA (1 g) and the total amount thereofwas 160 ml. The reaction was performed by anaerobically shaking at 37°C., 120 rpm for 36 hr in the presence of an oxygen adsorbent Anaeropack(Mitsubishi Chemical Corporation). After the reaction, 5N HCl (1.6 ml),chloroform (200 ml) and methanol (200 ml) were added to the reactionmixture (160 ml), the mixture was stirred with a stirrer, and thechloroform layer was recovered. Furthermore, chloroform (150 ml) wasadded to the residual solution, the mixture was stirred well, and thechloroform layer was recovered again. The recovered chloroform layerswere collectively concentrated in a rotary evaporator, and the reactionproduct and an unreacted substrate were extracted. A part of the extractwas methylesterified, and the purity of HYA was evaluated by gaschromatography. As a result, about 80% of the extract was confirmed tobe HYA.

Example 8

Purification of HYA from Extract (Mixture Containing HYA) Obtained inExample 7

Silica gel (Wakogel(R)C-100) in a 10-fold weight that of the extract(mixture containing HYA) obtained in Example 7 was swollen with hexaneand filled in a glass column, and sodium sulfate (anhydrous) was layeredthereon. The extract (mixture containing HYA) obtained in Example 7 wassuspended in an eluent of hexane:diethyl ether=8:2 and applied to thecolumn. The eluate was flown at a flow rate of about 2 ml, and thesolution discharged from the column was recovered by dividing intofractions. Each recovered fraction was analyzed by LC/MS and gaschromatography, unreacted substrate and fungus-derived lipid wereremoved, the eluent was changed to hexane:diethyl ether=6:4, and thefraction was further eluted. Each recovered fraction was analyzed byLC/MS and gas chromatography, and the fractions containing HYA only werecollected and concentrated in a rotary evaporator. A part of theobtained final product was methylesterified, and the purity of HYA wasevaluated by gas chromatography. As a result, HYA having a purity of notless than 98% was obtained.

Example 9

Production of KetoA from HYA by Using CLA-DH Expressed in Escherichiacoli

Using the purified dehydrogenase obtained in Example 6, a productiontest of KetoA from HYA was performed. The reaction mixture was 20 mMpotassium phosphate buffer (pH 8.0) containing purified dehydrogenase(enzyme amount 83 μg), 0.5 mM NAD⁺, 0.01 mM FAD and 0.1% HYA and thetotal amount thereof was 1 ml. The reaction was performed byanaerobically shaking at 37° C., 120 rpm for 4 hr in the presence of anoxygen adsorbent Anaeropack (Mitsubishi Chemical Corporation). After thereaction, lipid was extracted by Bligh-Dyer method from the reactionmixture and methylesterified, and the production of KetoA was evaluatedby gas chromatography. As a result, production of KetoA (0.06 mg) fromHYA was confirmed.

Example 10

Production of KetoA from HYA by Using Anhydrous Chromic Acid (CrO₃)

To anhydrous chromic acid (2.67 g) were added sulfuric acid (2.3 ml) andwater (7.7 ml), and acetone (90 ml) was added thereto to give a chromicacid solution. 2 g of HYA and 40 ml of acetone were added into anErlenmeyer flask, and the above-mentioned chromic acid solution wasadded drop by drop on ice while stirring the mixture with a stirrer.When the solution turned from blue to the color of powdered green tea,the dropwise addition of the chromic acid solution was stopped and thereaction was quenched with isopropyl alcohol. The precipitated sedimentwas filtered with a filter paper and placed in a partitioning funnel.Diethyl ether (150 ml) and Milli Q water (300 ml) were further added andthe mixture was shaken well. The diethyl ether layer was washed severaltimes with Milli Q water. To the diethyl ether layer after washing wasadded an appropriate amount of sodium sulfate (anhydrous), the mixturewas stirred, and the residual water was removed. The anhydrous sodiumsulfate added was filtered off with a filter paper, the obtained diethylether layer was concentrated in a rotary evaporator, and the reactionproduct and an unreacted substrate were extracted. Using a part of theextract, the purity of KetoA was evaluated by LC/MS. As a result, about95% of the extract was confirmed to be KetoA.

Example 11

Comparison of KetoA Production Using Anhydrous Chromic Acid and KetoAProduction Using CLA-DH Expressed in Escherichia coli

The conversion efficiency of the KetoA production method using purifieddehydrogenase was 6% since 0.06 mg of KetoA was produced from 1 ml ofreaction system containing 1 mg of HYA.

In contrast, the conversion efficiency of the KetoA production methodusing anhydrous chromic acid was about 95% since about 95% of the totalfatty acid extracted from 2 g of HYA was KetoA, and drastic improvementof KetoA production efficiency was successfully achieved.

Example 12

Purification of KetoA from Extract (Mixture Containing KetoA) Obtainedin Example 10

Silica gel (Wakogel(R)C-100) in a 20- to 30-fold weight that of theextract (mixture containing KetoA) obtained in Example 10 was swollenwith hexane and filled in a glass column, and sodium sulfate (anhydrous)was layered thereon. The extract (mixture containing KetoA) obtained inExample 10 was suspended in an eluent of hexane:diethyl ether=8:2 andapplied to the column. The eluent was flown at a flow rate of about 2ml, and the solution discharged from the column was recovered in dividedfractions. Each recovered fraction was analyzed by LC/MS and gaschromatography, and the fractions containing KetoA only were collectedand concentrated in a rotary evaporator. A part of the obtained finalproduct was methylesterified, and the purity of KetoA was evaluated bygas chromatography. As a result, KetoA having a purity of not less than98% was obtained.

Example 13

Production of αHYA from α-Linolenic Acid by Using Hydratase Expressed inEscherichia coli

Using hydratase-induced transformed Escherichia coli, a production testof αHYA from α-linolenic acid was performed. The reaction mixture was100 mM potassium phosphate buffer (pH 6.5) containing hydratase-inducedtransformed Escherichia coli (wet fungus weight 0.7 g), NADH (33 mg),FAD (0.8 mg), α-linolenic acid (1 g) and BSA (0.2 g) and the totalamount thereof was 10 ml. The reaction was performed by anaerobicallyshaking at 37° C., 225 rpm for 63 hr in the presence of an oxygenadsorbent Anaeropack (Mitsubishi Chemical Corporation). After thereaction, chloroform (10 ml) and methanol (10 ml) were added, themixture was stirred, and the chloroform layer was recovered.Furthermore, chloroform (10 ml) was added to the residual solution, themixture was stirred well, and the chloroform layer was recovered again.The recovered chloroform layers were collectively concentrated in arotary evaporator, and the reaction product and an unreacted substratewere extracted. A part of the extract was methylesterified, and thepurity of αHYA was evaluated by gas chromatography. As a result, about35% of the extract was confirmed to be αHYA.

Example 14

Purification of αHYA from Extract (Mixture Containing αHYA) Obtained inExample 13

Silica gel (Wakogel(R)C-100) in a 20- to 30-fold weight that of theextract (mixture containing αHYA) obtained in Example 13 was swollenwith hexane and filled in a glass column, and sodium sulfate (anhydrous)was layered thereon. The extract (mixture containing αHYA) obtained inExample 13 was suspended in an eluent of hexane:diethyl ether=8:2 andapplied to the column. The eluate was flown at a flow rate of about 3ml, and the solution discharged from the column was recovered in dividedfractions. Each recovered fraction was analyzed by LC/MS and gaschromatography, unreacted substrate and fungus-derived lipid wereremoved. The eluent was changed to hexane:diethyl ether=6:4, and thefraction was further eluted. Each recovered fraction was analyzed byLC/MS and gas chromatography, and the fractions containing αHYA onlywere collected and concentrated in a rotary evaporator. A part of theobtained final product was methylesterified, and the purity of αHYA wasevaluated by gas chromatography. As a result, αHYA having a purity ofnot less than 99% was obtained.

Example 15

Production of αKetoA from αHYA by Using Anhydrous Chromic Acid (CrO₃)

To anhydrous chromic acid (2.67 g) were added sulfuric acid (2.3 ml) andwater (7.7 ml), and acetone (90 ml) was added thereto to give a chromicacid solution. 2 g of αHYA and 40 ml of acetone were added into anErlenmeyer flask, and the above-mentioned chromic acid solution wasadded drop by drop on ice while stirring the mixture with a stirrer.When the solution turned from blue to the color of powdered green tea,the dropwise addition of the chromic acid solution was stopped and thereaction was quenched with isopropyl alcohol. The precipitated sedimentwas filtered with a filter paper and placed in a partitioning funnel.Diethyl ether (150 ml) and Milli Q water (300 ml) were further added andthe mixture was shaken well. The diethyl ether layer was washed severaltimes with Milli Q water. To the diethyl ether layer after washing wasadded an appropriate amount of sodium sulfate (anhydrous), the mixturewas stirred, and the residual water was removed. The anhydrous sodiumsulfate added was filtered off with a filter paper, the obtained diethylether layer was concentrated in a rotary evaporator, and the reactionproduct and an unreacted substrate were extracted. Using a part of theextract, the purity of αKetoA was evaluated by LC/MS. As a result, about80% of the extract was confirmed to be αKetoA.

Example 16

Purification of αKetoA from Extract (Mixture Containing αKetoA) Obtainedin Example 15

Silica gel (Wakogel(R)C-100) in a 20- to 30-fold weight that of theextract (mixture containing αKetoA) obtained in Example 15 was swollenwith hexane and filled in a glass column, and sodium sulfate (anhydrous)was layered thereon. The extract (mixture containing αKetoA) obtained inExample 15 was suspended in an eluent of hexane:diethyl ether=8:2 andapplied to the column. The eluent was flown at a flow rate of about 2ml, and the solution discharged from the column was recovered in dividedfractions. Each recovered fraction was analyzed by LC/MS and gaschromatography, and the fractions containing αKetoA only were collectedand concentrated in a rotary evaporator. A part of the obtained finalproduct was methylesterified, and the purity of αKetoA was evaluated bygas chromatography. As a result, αKetoA having a purity of not less than98% was obtained.

Example 17

Production of γHYA from γ-Linolenic Acid by Using Hydratase Expressed inEscherichia coli

Using hydratase-induced transformed Escherichia coli, a production testof γHYA from γ-linolenic acid was performed. The reaction mixture was100 mM potassium phosphate buffer (pH 6.5) containing hydratase-inducedtransformed Escherichia coli (wet fungus weight 0.7 g), NADH (33 mg),FAD (0.8 mg), γ-linolenic acid (1 g) and BSA (0.2 g) and the totalamount thereof was 10 ml. The reaction was performed by anaerobicallyshaking at 37° C., 225 rpm for 63 hr in the presence of an oxygenadsorbent Anaeropack (Mitsubishi Chemical Corporation). After thereaction, chloroform (10 ml) and methanol (10 ml) were added, themixture was stirred, and the chloroform layer was recovered.Furthermore, chloroform (10 ml) was added to the residual solution, themixture was stirred well, and the chloroform layer was recovered again.The recovered chloroform layers were collectively concentrated in arotary evaporator, and the reaction product and an unreacted substratewere extracted. A part of the extract was methylesterified, and thepurity of γHYA was evaluated by gas chromatography. As a result, about85% of the extract was confirmed to be γHYA.

Example 18

Purification of γHYA from Extract (Mixture Containing γHYA) Obtained inExample 17

Silica gel (Wakogel(R)C-100) in a 20- to 30-fold weight that of theextract (mixture containing γHYA) obtained in Example 17 was swollenwith hexane and filled in a glass column, and sodium sulfate (anhydrous)was layered thereon. The extract (mixture containing γHYA) obtained inExample 17 was suspended in an eluent of hexane:diethyl ether=8:2 andapplied to the column. The eluent was flown at a flow rate of about 3ml, and the solution discharged from the column was recovered in dividedfractions. Each recovered fraction was analyzed by LC/MS and gaschromatography, unreacted substrate and fungus-derived lipid wereremoved, the eluent was changed to hexane:diethyl ether=6:4, and thefraction was further eluted. Each recovered fraction was analyzed byLC/MS and gas chromatography, and the fractions containing γHYA onlywere collected and concentrated in a rotary evaporator. A part of theobtained final product was methylesterified, and the purity of γHYA wasevaluated by gas chromatography. As a result, γHYA having a purity ofnot less than 99% was obtained.

Example 19

Production of γKetoA from γHYA by Using Anhydrous Chromic Acid (CrO₃)

To anhydrous chromic acid (2.67 g) were added sulfuric acid (2.3 ml) andwater (7.7 ml), and acetone (90 ml) was added thereto to give a chromicacid solution. 2 g of γHYA and 40 ml of acetone were added into anErlenmeyer flask, and the above-mentioned chromic acid solution wasadded drop by drop on ice while stirring the mixture with a stirrer.When the solution turned from blue to the color of powdered green tea,the dropwise addition of the chromic acid solution was stopped and thereaction was quenched with isopropyl alcohol. The precipitated sedimentwas filtered with a filter paper and placed in a partitioning funnel.Diethyl ether (150 ml) and Milli Q water (300 ml) were further added andthe mixture was shaken well. The diethyl ether layer was washed severaltimes with Milli Q water. To the diethyl ether layer after washing wasadded an appropriate amount of sodium sulfate (anhydrous), the mixturewas stirred, and the residual water was removed. The anhydrous sodiumsulfate added was filtered off with a filter paper, the obtained diethylether layer was concentrated in a rotary evaporator, and the reactionproduct and an unreacted substrate were extracted. Using a part of theextract, the purity of γKetoA was evaluated by LC/MS. As a result, about95% of the extract was confirmed to be γKetoA.

Example 20

Production of sHYA from Stearidonic Acid by Using Hydratase Expressed inEscherichia coli

Using hydratase-induced transformed Escherichia coli, a production testof sHYA from stearidonic acid was performed. The reaction mixture was100 mM potassium phosphate buffer (pH 6.5) containing hydratase-inducedtransformed Escherichia coli (wet fungus weight 0.7 g), NADH (33 mg),FAD (0.8 mg), stearidonic acid (0.2 g) and BSA (40 mg) and the totalamount thereof was 10 ml. The reaction was performed by anaerobicallyshaking at 37° C., 225 rpm for 63 hr in the presence of an oxygenadsorbent Anaeropack (Mitsubishi Chemical Corporation). After thereaction, chloroform (10 ml) and methanol (10 ml) were added to thereaction mixture (160 ml), the mixture was stirred, and the chloroformlayer was recovered. Furthermore, chloroform (10 ml) was added to theresidual solution, the mixture was stirred well, and the chloroformlayer was recovered again. The recovered chloroform layers werecollectively concentrated in a rotary evaporator, and the reactionproduct and an unreacted substrate were extracted. A part of the extractwas methylesterified, and the purity of sHYA was evaluated by gaschromatography. As a result, about 50% of the extract was confirmed tobe sHYA.

Example 21

Production of 10,12-Dihydroxyoctadecanoic Acid (rHYA) from RicinoleicAcid by Using Hydratase Expressed in Escherichia coli

Using hydratase-induced transformed Escherichia coli, a production testof rHYA from ricinoleic acid was performed. The reaction mixture was 100mM potassium phosphate buffer (pH 6.5) containing hydratase-inducedtransformed Escherichia coli (wet fungus weight 0.7 g), NADH (33 mg),FAD (0.8 mg), ricinoleic acid (1 g) and BSA (0.2 g) and the total amountthereof was 10 ml. The reaction was performed by anaerobically shakingat 37° C., 225 rpm for 63 hr in the presence of an oxygen adsorbentAnaeropack (Mitsubishi Chemical Corporation). After the reaction,chloroform (10 ml) and methanol (10 ml) were added to the reactionmixture, the mixture was stirred, and the chloroform layer wasrecovered. Furthermore, chloroform (10 ml) was added to the residualsolution, the mixture was stirred well, and the chloroform layer wasrecovered again. The recovered chloroform layers were collectivelyconcentrated in a rotary evaporator, and the reaction product and anunreacted substrate were extracted. A part of the extract wasmethylesterified, and the purity of rHYA was evaluated by gaschromatography. As a result, about 95% of the extract was confirmed tobe rHYA.

Example 22

Production of KetoC from KetoA by Using Isomerase Expressed inEscherichia coli

KetoA (1 g), BSA (0.2 g) and 100 mM potassium phosphate buffer (pH 7.5,4 ml) were emulsified by ultrasonication, and the emulsion was dispensedinto 10 test tubes. To each test tube was added isomerase-expressingtransformed Escherichia coli, suspended in 100 mM potassium phosphatebuffer (pH 7.5) at 2 ml/g, to the total amount of 1 ml. The reaction wasperformed by anaerobically shaking at 37° C., 225 rpm for 15 hr in thepresence of an oxygen adsorbent Anaeropack (Mitsubishi ChemicalCorporation). After the reaction, 2 ml of methanol was added to eachtest tube, the mixture was stirred by Vortex and, after centrifugation,the supernatant was recovered. Furthermore, 2 ml of methanol was addedto the residue, the mixture was stirred by Vortex and, aftercentrifugation, the supernatant was recovered again. The recoveredsupernatants were collectively concentrated in a rotary evaporator. 1 mlof distilled water and 3 ml of hexane were added to the concentrate, themixture was stirred by Vortex, and the hexane layer was recovered aftercentrifugation. The reaction product and an unreacted substrate wereextracted. Furthermore, 3 ml of hexane was added to the residualsolution, the mixture was stirred well, and the hexane layer wasrecovered again after centrifugation. The recovered hexane layers werecollectively concentrated in a rotary evaporator, and the reactionproduct and an unreacted substrate were extracted. A part of the extractwas methylesterified, and the purity of KetoC was evaluated by gaschromatography. As a result, about 56% of the extract was confirmed tobe KetoC.

Example 23

Purification of KetoC by HPLC

Monitoring was performed by using Develosil C30-UG-3 manufactured byNOMURA CHEMICAL CO., LTD. (10×150 mm), mobile phase ofacetonitrile:water:acetic acid (80:20:0.002), flow rate 3.5 ml/min,column temperature 30° C., detection by absorption at 225 nm. Themixture obtained in the above-mentioned Example 24 was dissolved inmethanol at 100 mg/ml, and 0.15 ml was applied to the column. Only thepeak of KetoC eluted at retention time about 7.5 min was fractionated.The eluents in the fractionated solutions were collectively removed inan evaporator. A part of the obtained final product wasmethylesterified, and the purity of KetoC was evaluated by gaschromatography and LC/MS. As a result, KetoC was obtained at a purity ofnot less than 98%.

Example 24

Production of αKetoC from αKetoA by Using Isomerase Expressed inEscherichia coli

αKetoA (0.5 g), BSA (0.1 g) and 100 mM potassium phosphate buffer (pH7.5, 2 ml) were emulsified by ultrasonication, and the emulsion wasdispensed into 4 test tubes. To each test tube was addedisomerase-expressing transformed Escherichia coli (0.5 ml), suspended in100 mM potassium phosphate buffer (pH 7.5) at 2 ml/g, to the totalamount of 1 ml. The reaction was performed by anaerobically shaking at37° C., 225 rpm for 15 hr in the presence of an oxygen adsorbentAnaeropack (Mitsubishi Chemical Corporation). After the reaction, 2 mlof methanol was added to each test tube, the mixture was stirred byVortex and, after centrifugation, the supernatant was recovered.Furthermore, 2 ml of methanol was added to the residue, the mixture wasstirred by Vortex and, after centrifugation, the supernatant wasrecovered again. The recovered supernatants were collectivelyconcentrated in a rotary evaporator. 1 ml of distilled water and 3 ml ofhexane were added to the concentrate, the mixture was stirred by Vortex,and the hexane layer was recovered after centrifugation. The reactionproduct and an unreacted substrate were extracted. Furthermore, 3 ml ofhexane was added to the residual solution, the mixture was stirred well,and the hexane layer was recovered again after centrifugation. Therecovered hexane layers were collectively concentrated in a rotaryevaporator, and the reaction product and an unreacted substrate wereextracted. Using a part of the extract, the purity of αKetoC wasevaluated by high performance liquid chromatography. As a result, about65% of the extract was confirmed to be αKetoC.

Example 25

Purification of αKetoC by HPLC

Monitoring was performed by using Develosil C30-UG-5 manufactured byNOMURA CHEMICAL CO., LTD., mobile phase of acetonitrile:water:aceticacid (60:40:0.002), flow rate 10 ml/min, column temperature 30° C.,detection by absorption at 210 nm and 233 nm. The mixture obtained inthe above-mentioned Example 26 was dissolved in methanol at 100 mg/ml,and 0.17 ml was applied to the column. Only the peak of αKetoC elutedwas fractionated by using recycling system. The eluents in thefractionated solutions were collectively removed in an evaporator. Apart of the obtained final product was methylesterified, and the purityof αKetoC was evaluated by gas chromatography and LC/MS. As a result,αKetoC was obtained at a purity of not less than 98%.

Example 26

Production of γKetoC from γKetoA by Using Isomerase Expressed inEscherichia coli

γKetoA (0.5 g), BSA (0.1 g) and 100 mM potassium phosphate buffer (pH7.5, 2 ml) were emulsified by ultrasonication, and the emulsion wasdispensed into 4 test tubes. To each test tube was addedisomerase-expressing transformed Escherichia coli (each 0.5 ml),suspended in 100 mM potassium phosphate buffer (pH 7.5) at 2 ml/g, tothe total amount of 1 ml. The reaction was performed by anaerobicallyshaking at 37° C., 225 rpm for 15 hr in the presence of an oxygenadsorbent Anaeropack (Mitsubishi Chemical Corporation). After thereaction, 2 ml of methanol was added to each test tube, the mixture wasstirred by Vortex and, after centrifugation, the supernatant wasrecovered. Furthermore, 2 ml of methanol was added to the residue, themixture was stirred by Vortex and, after centrifugation, the supernatantwas recovered again. The recovered supernatants were collectivelyconcentrated in a rotary evaporator. 1 ml of distilled water and 3 ml ofhexane were added to the concentrate, the mixture was stirred by Vortex,and the hexane layer was recovered after centrifugation. The reactionproduct and an unreacted substrate were extracted. Furthermore, 3 ml ofhexane was added to the residual solution, the mixture was stirred well,and the hexane layer was recovered again after centrifugation. Therecovered hexane layers were collectively concentrated in a rotaryevaporator, and the reaction product and an unreacted substrate wereextracted. Using a part of the extract, the purity of γKetoC wasevaluated by LC/MS. As a result, about 95% of the extract was confirmedto be γKetoC.

Example 27

Purification of γKetoC by HPLC

Monitoring was performed by using Develosil C30-UG-5 manufactured byNOMURA CHEMICAL CO., LTD., mobile phase of acetonitrile:water:aceticacid (60:40:0.002), flow rate 10 ml/min, column temperature 30° C.,detection by absorption at 210 nm and 233 nm. The mixture obtained inthe above-mentioned Example 26 was dissolved in methanol at 100 mg/ml,and 0.17 ml was applied to the column. Only the peak of γKetoC elutedwas fractionated by using recycling system. The eluents in thefractionated solutions were collectively removed in an evaporator. Apart of the obtained final product was methylesterified, and the purityof γKetoC was evaluated by gas chromatography and LC/MS. As a result,γKetoC was obtained at a purity of not less than 96%.

Example 28

Production of KetoC from KetoA by Using Isomerase Expressed inEscherichia coli

KetoA (1 g), BSA (0.2 g) and 100 mM potassium phosphate buffer (pH 7.5,4 ml) were emulsified by ultrasonication, and the emulsion was dispensedinto 10 test tubes. To each test tube was added isomerase-expressingtransformed Escherichia coli, suspended in 100 mM potassium phosphatebuffer (pH 7.5) at 2 ml/g, to the total amount of 1 ml. The reaction wasperformed by anaerobically shaking at 37° C., 225 rpm for 15 hr in thepresence of an oxygen adsorbent Anaeropack (Mitsubishi ChemicalCorporation). After the reaction, 2 ml of methanol was added to eachtest tube, the mixture was stirred by Vortex and, after centrifugation,the supernatant was recovered. Furthermore, 2 ml of methanol was addedto the residue, the mixture was stirred by Vortex and, aftercentrifugation, the supernatant was recovered again. The recoveredsupernatants were collectively concentrated in a rotary evaporator. 1 mlof distilled water and 3 ml of hexane were added to the concentrate, themixture was stirred by Vortex, and the hexane layer was recovered aftercentrifugation. The reaction product and an unreacted substrate wereextracted. Furthermore, 3 ml of hexane was added to the residualsolution, the mixture was stirred well, and the hexane layer wasrecovered again after centrifugation. The recovered hexane layers werecollectively concentrated in a rotary evaporator, and the reactionproduct and an unreacted substrate were extracted. A part of the extractwas methylesterified, and the purity of KetoC was evaluated by gaschromatography. As a result, about 56% of the extract was confirmed tobe KetoC.

Example 29

Purification of KetoC by HPLC

Monitoring was performed by using Develosil C30-UG-3 manufactured byNOMURA CHEMICAL CO., LTD. (10×150 mm), mobile phase ofacetonitrile:water:acetic acid (80:20:0.002), flow rate 3.5 ml/min,column temperature 30° C., detection by absorption at 225 nm. Themixture obtained in the above-mentioned Example 28 was dissolved inmethanol at 100 mg/ml, and 0.15 ml was applied to the column. Only thepeak of KetoC eluted at retention time about 7.5 min was fractionated.The eluents in the fractionated solutions were collectively removed inan evaporator. A part of the obtained final product wasmethylesterified, and the purity of KetoC was evaluated by gaschromatography and LC/MS. As a result, KetoC was obtained at a purity ofnot less than 98%.

Example 30

Production of KetoB from KetoC by Using Saturase Expressed inEscherichia coli

Using saturase, a production test of KetoB was performed. The reactionmixture was 100 mM potassium phosphate buffer (pH 6.5) containingsaturase-induced transformed Escherichia coli (wet fungus weight 33 mg),0.5 mM NADH, 0.01 mM FAD, KetoC (5.2 mg) and 1 mg of BSA, and the totalamount thereof was 1 ml. The reaction was performed by anaerobicallyshaking at 37° C., 200 rpm for 17 hr in the presence of an oxygenadsorbent Anaeropack (Mitsubishi Chemical Corporation). After thereaction, lipid was extracted from the reaction mixture by theBligh-Dyer method and methylesterified, after which the production ofKetoB was evaluated by gas chromatography. As a result, it was clarifiedthat KetoB was produced at a conversion rate of 30%.

Example 31

Production of αKetoB from αKetoA by Using Isomerase and SaturaseExpressed in Escherichia coli

Using isomerase and saturase, a production test of αKetoB was performed.The reaction mixture was 100 mM potassium phosphate buffer (pH 7.5)containing isomerase-induced transformed Escherichia coli (wet fungusweight 80 mg), saturase-induced transformed Escherichia coli (wet fungusweight 80 mg), 0.5 mM NADH, 0.01 mM FAD, αKetoA (2 mg) and 0.4 mg BSA,and the total amount thereof was 1 ml. The reaction was performed byanaerobically shaking at 37° C., 225 rpm for 18 hr in the presence of anoxygen adsorbent Anaeropack (Mitsubishi Chemical Corporation). After thereaction, lipid was extracted from the reaction mixture by theBligh-Dyer method and methylesterified, after which the production ofαKetoB was evaluated by gas chromatography. As a result, it wasclarified that αKetoB was produced at a conversion rate of 99%.

Example 32

Production of HYC from KetoC by Using Dehydrogenase Expressed inEscherichia coli

Using the purified dehydrogenase obtained in Example 6, a productiontest of HYC was performed. The reaction mixture was 20 mM potassiumphosphate buffer (pH 6.5) containing purified dehydrogenase (enzymecontent 83 μg), 0.5 mM NADH, 0.01 mM FAD and 0.005% KetoC and the totalamount thereof was 0.8 ml. The reaction was performed by anaerobicallyshaking at 37° C., 120 rpm for 4 hr in the presence of an oxygenadsorbent Anaeropack (Mitsubishi Chemical Corporation). After thereaction, lipid was extracted from the reaction mixture by theBligh-Dyer method and methylesterified, after which the production ofHYC was evaluated by gas chromatography. As a result, production of HYC(0.02 mg) from KetoC was confirmed.

Example 33

Production of HYB from KetoB by Using Dehydrogenase Expressed inEscherichia coli

Using dehydrogenase, a production test of HYB was performed. Thereaction mixture was 20 mM potassium phosphate buffer (pH 6.5)containing dehydrogenase-induced transformed Escherichia coli (wetfungus weight 50 mg), 0.5 mM NADH, 0.01 mM FAD and 0.02% KetoB and thetotal amount thereof was 1 ml. The reaction was performed byanaerobically shaking at 37° C., 120 rpm for 4 hr in the presence of anoxygen adsorbent Anaeropack (Mitsubishi Chemical Corporation). After thereaction, lipid was extracted from the reaction mixture by theBligh-Dyer method and methylesterified, after which the production ofHYB was evaluated by gas chromatography. As a result, production of HYB(0.08 mg) from KetoB was confirmed.

Example 34

Production of CLA1 and CLA2 from HYC by Using Dehydrase Expressed inEscherichia coli

Using the purified dehydrase obtained in Example 5, a production test ofCLA1 and CLA2 was performed. The reaction mixture was 20 mM potassiumphosphate buffer (pH 6.5) containing purified dehydrase (enzyme content300 μg), 0.5 mM NADH, 0.01 mM FAD and 0.035% HYC and the total amountthereof was 0.8 ml. The reaction was performed by anaerobically shakingat 37° C., 120 rpm for 4 hr in the presence of an oxygen adsorbentAnaeropack (Mitsubishi Chemical Corporation). After the reaction, lipidwas extracted from the reaction mixture by the Bligh-Dyer method andmethylesterified, after which the production of CLA1 and CLA2 wasevaluated by gas chromatography. As a result, production of CLA1 (0.05mg) and CLA2 (0.15 mg) from HYC was confirmed.

Example 35

Production of Oleic Acid and Trans-10-Octadecenoic Acid from HYB byUsing Dehydrase Expressed in Escherichia coli

Using dehydrase, a production test of oleic acid andtrans-10-octadecenoic acid was performed. The reaction mixture was 20 mMpotassium phosphate buffer (pH 6.5) containing dehydrase-inducedtransformed Escherichia coli (wet fungus 50 mg), 0.5 mM NADH, 0.01 mMFAD and 0.2% HYB and the total amount thereof was 1 ml. The reaction wasperformed by anaerobically shaking at 37° C., 120 rpm for 4 hr in thepresence of an oxygen adsorbent Anaeropack (Mitsubishi ChemicalCorporation). After the reaction, lipid was extracted from the reactionmixture by the Bligh-Dyer method and methylesterified, after which theproduction of oleic acid and trans-10-octadecenoic acid was evaluated bygas chromatography. As a result, production of oleic acid (0.02 mg) andtrans-10-octadecenoic acid (0.1 mg) from HYB was confirmed.

Example 36

Production of Linoleic Acid and CLA3 from HYA by Using DehydraseExpressed in Escherichia coli

Using dehydrase, a production test of linoleic acid andtrans-10,cis-12-CLA was performed. The reaction mixture was 20 mMpotassium phosphate buffer (pH 6.5) containing dehydrogenase-inducedtransformed Escherichia coli (wet fungus 50 mg), 0.5 mM NADH, 0.01 mMFAD and 0.07% HYA and the total amount thereof was 1 ml. The reactionwas performed by anaerobically shaking at 37° C., 120 rpm for 4 hr inthe presence of an oxygen adsorbent Anaeropack (Mitsubishi ChemicalCorporation). After the reaction, lipid was extracted from the reactionmixture by the Bligh-Dyer method and methylesterified, after which theproduction of linoleic acid and CLA3 was evaluated by gaschromatography. As a result, production of linoleic acid (0.26 mg) andtrans-10,cis-12-CLA (0.07 mg) from HYA was confirmed.

While the present invention has been described with emphasis onpreferred embodiments, it is obvious to those skilled in the art thatthe preferred embodiments can be modified.

The contents disclosed in any publication cited herein, includingpatents and patent applications, are hereby incorporated in theirentireties by reference, to the extent that they have been disclosedherein.

This application is based on a patent application No. 2012-108928 filedin Japan (filing date: May 10, 2012), the contents of which areincorporated in full herein.

INDUSTRIAL APPLICABILITY

According to the method of the present invention, since various oxofatty acids can be produced efficiently, the oxo fatty acids can beapplied to various fields such as medicament, food and the like.According to the method of the present invention, moreover, various rarefatty acids can be produced from oxo fatty acid as a starting material,which is extremely useful.

The invention claimed is:
 1. A method of producing an oxo fatty acid having 18 carbon atoms, a carbonyl group at the 10-position and a trans-type double bond at the 11-position, comprising contacting an oxo fatty acid having 18 carbon atoms, a carbonyl group at the 10-position and a cis-type double bond at the 12-position with an isomerase, and recovering the resultant oxo fatty acid having 18 carbon atoms, a carbonyl group at the 10-position and a trans-type double bond at the 11-position, wherein the isomerase is obtained from Lactobacillus plantarum FERM BP-10549 strain, or a recombinant of the isomerase.
 2. The method according to claim 1, wherein the oxo fatty acid having 18 carbon atoms, a carbonyl group at the 10-position and a cis-type double bond at the 12-position is 10-oxo-cis-12-octadecenoic acid, 10-oxo-cis-6,cis-12-octadecadienoic acid, 10-oxo-cis-12,cis-15-octadecadienoic acid or 10-oxo-cis-6,cis-12,cis-15-octadecatrienoic acid. 